This study was aimed at establishing a method of ultra-fast quantitative PCR for Brucella detection.We used an exogenous recombinant plasmid as the internal reference and targeted the T4SS secretion system,an important Brucella viru-lence factor,to design specific primers and probes.The sensitivity,specificity,and repeatability of this method were evaluated,and a standard curve was constructed.The coincidence rate of detection findings with this method versus quantitative PCR was determined.This method markedly decreased the detection time to only 10 minutes.The standard curve demonstrated a good linear relationship(Y=-3.410 7x+38.357,R2=0.998 5)with a low minimum detection limit of 10 copies/μL.The method exhibited good specificity and did not specifically amplify several common clinical bacteria other than Brucella.The de-tection of three concentrations of positive plasmids yielded coefficients of variation(CVs)of 0.20％to 0.91％,thus demonstra-ting the method's excellent repeatability.Furthermore,140 clinical samples were analyzed concurrently with the fluorescence PCR method,which yielded a 100％compliance rate and consistent results.Our findings indicated that the Brucella ultra-fast quantitative PCR was ultrafast;had high sensitivity,high specificity,and good specificity;and can be used for the clinical de-tection of Brucella and emergency investigation of epidemics.Therefore,this method is valuable for the early diagnosis of Bru-cella.

This study was aimed at establishing a method of ultra-fast quantitative PCR for Brucella detection.We used an exogenous recombinant plasmid as the internal reference and targeted the T4SS secretion system,an important Brucella viru-lence factor,to design specific primers and probes.The sensitivity,specificity,and repeatability of this method were evaluated,and a standard curve was constructed.The coincidence rate of detection findings with this method versus quantitative PCR was determined.This method markedly decreased the detection time to only 10 minutes.The standard curve demonstrated a good linear relationship(Y=-3.410 7x+38.357,R2=0.998 5)with a low minimum detection limit of 10 copies/μL.The method exhibited good specificity and did not specifically amplify several common clinical bacteria other than Brucella.The de-tection of three concentrations of positive plasmids yielded coefficients of variation(CVs)of 0.20％to 0.91％,thus demonstra-ting the method's excellent repeatability.Furthermore,140 clinical samples were analyzed concurrently with the fluorescence PCR method,which yielded a 100％compliance rate and consistent results.Our findings indicated that the Brucella ultra-fast quantitative PCR was ultrafast;had high sensitivity,high specificity,and good specificity;and can be used for the clinical de-tection of Brucella and emergency investigation of epidemics.Therefore,this method is valuable for the early diagnosis of Bru-cella.

The NiFe2O4-graphene oxide nanocomposite modified with polyethylenimine(GO@PEI-NiFe2O4)was prepared to purify and enrich phosphopeptides from biosamples.The Ni2+and Fe3+ions on its surface could coordinate with phosphate groups and then selectively adsorb phosphopeptides.PEI was conducive to the above combination due to its high hydrophilicity.The material showed good magnetic response properties and could be rapidly separated from samples with the aid of magnet.With tryptic digest of β-casein as sample,the enrichment property of the material to phosphopeptides was studied,which was compared with the results of GO@NiFe2O4,revealing the adsorption mechanism of GO@PEI-NiFe2O4.The static and dynamic binding properties of GO@PEI-NiFe2O4 were investigated using pTyr as a representative phosphopeptide,and the adsorption capacity was 36.2 μg/mg.The results showed that the material could remove the interference of nonphosphopeptides and effectively enrich phosphopeptides in complex matrix.After enrichment by GO@PEI-NiFe2O4,1535 phosphopeptides were identified from the tryptic digest of rat liver by mass spectrum and the enrichment effect of GO@PEI-NiFe2O4 greatly outperformed commercial Fe3+-IMAC kits.This work provided an efficient material for the enrichment of phosphopeptides,showing potential applications in phosphoproteomics research.

OBJECTIVE: To investigate a family with congenital dysfibrinogenemia, and analyze the risk of hemorrhage and thrombosis and blood transfusion strategies. METHODS: Prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) of the proband and her family members were detected by automatic coagulometer, fibrinogen (Fg) activity and antigen were detected by Clauss method and PT algorithm respectively. Meanwhile, thromboelastometry was analyzed for proband and her family members. Then, peripheral blood samples of the proband and her family members were collected, and all exons of FGA, FGB and FGG and their flanks were amplified by PCR and sequenced to search for gene mutations. RESULTS: The proband had normal APTT and PT, slightly prolonged TT, reduced level of Fg activity (Clauss method). The Fg of the proband's aunt, son and daughter all decreased to varying degrees. The results of thromboelastogram indicated that Fg function of the proband and her family members (except her son) was basically normal. Gene analysis showed that there were 6233 G/A (p.AαArg35His) heterozygous mutations in exon 2 of FGA gene in the proband, her children and aunt. In addition, 2 polymorphic loci were found in the family, they were FGA gene g.9308A/G (p.AαThr331Ala) and FGB gene g.12628G/A (p.BβArg478Iys) polymorphism, respectively. The proband was injected with 10 units of cryoprecipitate 2 hours before delivery to prevent bleeding, and no obvious bleeding occurred during and after delivery. CONCLUSION Heterozygous mutation of 6233G/A (p.AαArg35His) of FGA gene is the biogenetic basis of the disease in this family with congenital dysfibrinogenemia.
Humans ; Child ; Female ; Fibrinogen/genetics* ; Pedigree ; Afibrinogenemia/genetics* ; Mutation ; Blood Transfusion

Humans ; Consensus ; Hypersensitivity ; Desensitization, Immunologic/adverse effects* ; Immunotherapy/adverse effects* ; Injections, Subcutaneous ; Allergens

Objective: To explore the effects of expanded frontal-parietal pedicled flap in reconstructing cervical scar contracture deformity in children after burns. Methods: A retrospective observational study was conducted. From January 2015 to December 2020, 18 male children with cervical scar contracture deformity after burns who met the inclusion criteria were admitted to Zhengzhou First People's Hospital, aged 4 to 12 years, including 10 cases with degree Ⅱ cervical scar contracture deformity and 8 cases with degree Ⅲ scar contracture deformity, and were all reconstructed with expanded frontal-parietal pedicled flap. The surgery was performed in 3 stages. In the first stage, a cylindrical skin and soft tissue expander (hereinafter referred to as expander) with rated capacity of 300 to 500 mL was placed in the frontal-parietal region. The expansion time was 4 to 6 months with the total normal saline injection volume being 2.1 to 3.0 times of the rated capacity of expander. In the second stage, expander removal, scar excision, contracture release, and flap transfer were performed, with the flap areas of 18 cm×9 cm to 23 cm×13 cm and the secondary wound areas of 16 cm×8 cm to 21 cm×11 cm after scar excision and contracture release. After 3 to 4 weeks, in the third stage, the flap pedicle was cut off and restored. The rated volume of placed expander, total normal saline injection volume, type of vascular pedicle of flap, survival of flap and reconstruction of scar after the second stage surgery were recorded. The neck range of motion and cervico-mental angle were measured before surgery and one-year after surgery. The appearance of neck, occurrence of common complications in the donor and recipient sites of children, and satisfaction of children's families for treatment effects were followed up. Data were statistically analyzed with paired sample t test. Results: All the patients successfully completed the three stages of operation. The rated volume of implanted expander was 300 mL in 6 children, 400 mL in 9 children, and 500 mL in 3 children, with the volume of normal saline injection being 630 to 1 500 mL. The type of vascular pedicle of flap was double pedicle in 13 cases and was single pedicle in 5 cases. All the flaps in 17 children survived well, and the secondary wounds after neck scar excision and contracture release were all reconstructed in one procedure. In one case, the distal blood supply of the single pedicled flap was poor after the second stage surgery, with necrosis of about 2.5 cm in length. The distal necrotic tissue was removed on 10 days after the operation, and the wound was completely closed after the flap was repositioned. In the follow-up of 6 months to 3 years post operation, the cervical scar contracture deformity in 18 children was corrected without recurrence. The flap was not bloated, the texture was soft, and the appearances of chin and neck were good. The range of motion of cervical pre-buckling, extension, left flexion, and right flexion, and cervico-mental angle in one year after operation were improved compared with those before operation (with t values of 43.10, 22.64, 27.96, 20.59, and 88.42, respectively, P<0.01). The incision in the frontal donor site was located in the hairline, the scar was slight and concealed. No complication such as cranial depression was observed in expander placement site, and the children's families were satisfied with the result of reconstruction. Conclusions: Application of expanded frontal-parietal pedicled flap in reconstructing the cervical scar contracture deformity in children after burns can obviously improve the appearance and function of neck, with unlikely recurrence of postoperative scar contractures, thus it is an ideal method of reconstruction.
Burns/surgery* ; Child ; Cicatrix/surgery* ; Contracture/surgery* ; Humans ; Male ; Perforator Flap ; Reconstructive Surgical Procedures/methods* ; Saline Solution ; Skin Transplantation ; Treatment Outcome

Objective: To explore the effect of three interventions including caloric restriction, rope-skipping exercise and caloric restriction combined with rope-skipping exercise on cardiometabolic risk factors in overweight or obese college students. Methods: This study was a pilot randomized controlled trial. Overweight or obese students who met the inclusion criteria were recruited at Sun Yat-sen University in September 2019 and were randomly divided into four groups: caloric restriction group (CR), rope-skipping group (RS), combined group (CR-RS) and control group (CT). The intervention in each group lasted 8 weeks, specifically: the daily energy intake of CR was 100% to 110% of the basal metabolic energy; RS was instructed to rope three times a week, and CR-RS combined caloric restriction with rope-skipping. At the baseline and end of 8-week intervention, basic information, anthropometric indicators and fasting vein blood of students were collected. Paired t test and Wilcoxon paired-samples signed rank test were used for comparison before and after intervention, and analysis of covariance was used for comparison between groups after intervention. Results: A total of 29 students completed the trial and were included in the final analysis (7, 9, 7 and 6 students in CR, RS, CR-RS and CT, respectively). The mean age of students were (19.00±1.00) years, including 11 males and 18 females. The baseline characteristics of four groups were comparable. After 8 weeks of intervention, compared with CT, there was an increase in the body fat percentage and fat mass index in CR and CR-RS (P<0.05). Insulin level decreased in CR-RS (P<0.05). Systolic blood pressure in CR and diastolic blood pressure in CR-RS were higher (P<0.05). Compared with baseline, fat mass index decreased in CR (P<0.05), while body weight, BMI, and fat mass index decreased in CR-RS (P<0.05). Conclusion: It is suggested that the caloric restriction alone and calorie restriction combined with rope-skipping exercise can benefit overweight or obese college students with cardiometabolic risk factors.
Adolescent ; Adult ; Caloric Restriction ; Cardiometabolic Risk Factors ; Female ; Humans ; Insulins ; Male ; Obesity ; Overweight ; Students ; Weight Loss/physiology* ; Young Adult

Objective: To evaluate the value of the 2020 diagnostic criteria (Cellucci criteria) for pediatric autoimmune encephalitis (AE) in children with suspected AE in a single center. Methods: The clinical data of 121 children hospitalized at the First Affiliated Hospital of Zhengzhou University from October 2019 to October 2021, with a diagnosis of suspected AE, were retrospectively collected and analyzed. The children were divided into definite antibody-positive AE (dAPAE), probable antibody-negative AE (prANAE), possible AE (pAE) and non-AE groups according to the Chinese expert consensus and the Graus criteria. A new diagnosis was made according to the Cellucci criteria which was compared with the clinical diagnosis to evaluate the diagnostic value of the Cellucci criteria. The Mann-Whitney U test, Kruskal-Wallis test, and χ² test were used to compare the differences among groups. The sensitivity and specificity were used to evaluate efficacy of the Cellucci criteria. Results: Among the 121 children, 72 were males and 49 were females, with an age of 10.3 (6.5, 14.0) years at disease onset. There were 99 cases diagnosed as AE according the clinical diagnosis (58 males and 41 females), of which 43 cases were diagnosed as dAPAE, 14 cases as prANAE and 42 cases as pAE, and the other 22 cases were not AE (14 males and 8 females). The top 2 initial symptoms in the 99 children with AE were seizures (53 cases, 53.5%) and abnormal mental behaviors (35 cases, 35.4%). And the most common symptoms during the course of the disease were abnormal mental behaviors (77 cases, 77.8%) and seizures (64 cases, 64.6%). There were statistically differences in the incidence of consciousness disorders, autonomic dysfunctions during the course of the disease and the length of hospitalization among the 4 groups (χ²=21.63, 13.74, H=22.60, all P<0.05). Ninety-six of the 121 children were tested for AE-related antibodies, of which 45 cases (46.9%) were antibody-positive. According to the Cellucci criteria, 42 cases were diagnosed as dAPAE, 34 cases as prANAE and 14 cases as pAE. Compared with the clinical diagnosis, the sensitivity of the Cellucci criteria for the diagnosis of the 3 types of AE were 93.02%, 92.86% and 87.88%, and the specificity were 96.23%, 74.39% and 86.36%, respectively. Conclusions: The Cellucci criteria has a high sensitivity and specificity for the diagnosis of pAE and dAPAE in the clinical management of children with suspected AE, while a high sensitivity but low specificity for the diagnosis of prANAE. Therefore, it is recommended to apply the Cellucci criteria selectively in clinical practice according to the actual situation, especially in the diagnosis of prANAE.
Child ; Encephalitis/diagnosis* ; Female ; Hashimoto Disease/diagnosis* ; Humans ; Male ; Retrospective Studies ; Seizures

Objective: To explore the effect of dormant polyploid giant cancer cells (PGCC) on nasopharyngeal carcinoma (NPC) recurrence and to clarify the role of inhibition of autophagy in inhibiting NPC-PGCC formation and preventing NPC recurrence. Methods: NPC cells-derived PGCC (NPC-PGCC) were induced by paclitaxel (PTX), and the morphology, polyploid characteristics and cell activity of PGCC were identified by light microscopy, immunofluorescence and Live/Dead cell double staining assays. RNA-seq was used to analyze the differentially expressed genes between NPC-PGCC and diploid nasopharyngeal carcinoma cells CNE2. Functional enrichment and pathway annotation analysis of differentially expressed genes were performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG). The level of autophagy in NPC-PGCC cells was assessed by Western Blot and transmission electron microscopy analysis. The role of autophagy in the formation of NPC-PGCC and the effect of NPC-PGCC on the recurrence of nasopharyngeal carcinoma were studied using a highly clinically relevant mouse nasopharyngeal carcinoma recurrence model. Statistical analysis was performed using GraphPad Prism 6 and P-values<0.05 were considered statistically significant. Results: NPC-PGCC induced by paclitaxel had the characteristics of burst-like division after dormancy. GO enrichment and KEGG pathway analyses identified the significant biological processes and pathways mainly concentrated in autophagy and related pathways involving the differentially expressed genes between NPC-PGCC and diploid nasopharyngeal carcinoma cells CNE2. The autophagy level was significantly enhanced in NPC-PGCC cells. In a highly clinically relevant mouse nasopharyngeal carcinoma recurrence model, the number of PGCC in the primary tumor of the nude mice treated with cisplatin were higher than those of the other groups. In nude mice pretreated with autophagy inhibitor and then co-treatment with autophagy inhibitor and cisplatin, the number of PGCC in primary tumors was less and the recurrence rate was significantly lower than in other groups. Conclusions: The mechanism of dormant polyploid giant cancer cells formation is related to autophagy. Inhibition of autophagy can inhibit the formation of PGCC and thus prevent the recurrence of nasopharyngeal carcinoma.
Animals ; Autophagy ; Carcinoma/genetics* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Cisplatin/pharmacology* ; Gene Expression Regulation, Neoplastic ; Mice ; Mice, Nude ; Nasopharyngeal Carcinoma/genetics* ; Nasopharyngeal Neoplasms/pathology* ; Paclitaxel/pharmacology* ; Polyploidy

Objective: To investigate the molecular pathogenesis and clinical features of unrelated 12 patients with inherited coagulation protein C (PC) deficiency in Chinese population. Methods: The PC activity (PC:A) and PC antigen (PC:Ag) were detected by chromogenic substrate and enzyme linked immunosorbent assay, respectively. The nine exons and flanking sequences of the protein C (PROC) gene were amplified by polymerase chain reaction with direct sequencing, and the suspected mutations were validated by reverse sequencing (clone sequencing for deletion mutations) . Results: The PC:A of the 12 probands decreased significantly, ranging from 18% to 55%, and the PC:Ag of the 10 probands decreased significantly. Eleven mutations were found, out of which four mutations [c.383G>A (p.Gly128Asp) , c.997G>A (p.Ala291Thr) , c.1318C>T (p.Arg398Cys) , and c.532G>C (p.Leu278Pro) ] were discovered for the first time. Six mutations were in the serine protease domain, four mutations were located in epidermal growth factor (EGF) -like domains, and one mutation was located in activation peptide. There were two deletion mutations (p.Met364Trp fsX15 and p.Lys192del) , and the rest were missense mutations. Mutations p.Phe181Val and p.Arg189Trp were identified in three unrelated families. All mutations may be inherited, and consanguineous marriages were reported in two families. Among the probands, nine cases had venous thrombosis, two cases had poor pregnancy manifestations, and one case had purpura. Conclusion: Patients with PC deficiency caused by PROC gene defects are prone to venous thrombosis, especially when there are other thrombotic factors present at the same time.
Humans ; Mutation ; Mutation, Missense ; Pedigree ; Phenotype ; Protein C/genetics* ; Protein C Deficiency/genetics*